Anti-Candida Antibodies of Patients with Invasive Candidiasis Inhibit Growth, Alter Cell Wall Structure, and Kill Candida albicans In Vitro

Abstract

Invasive candidiasis (IC), caused by Candida yeasts, particularly Candida albicans, poses a significant threat with high mortality rates. Diagnosis is challenging due to Candida's common presence in human microbiota. To address this, our research group developed an immunofluorescence assay detecting Candida albicans Germ Tube Antibodies (CAGTA) in IC patients. CAGTA, indicative of invasive processes, is associated with a lower mortality rate in ICU patients. Based on this premise, this study aims to provide results regarding the lack of knowledge about the potential activity of CAGTA against invasive infections in humans caused by the fungus Candida albicans. Therefore, in order to characterize the activity of CAGTA produced by patients with IC, we used sera from 29 patients with IC caused by either C. albicans or non-albicans Candida species. Whole serum IgG antibodies were fractionated into anti-blastospores, CAGTA-enriched, and purified CAGTA and the assessments included XTT colorimetric assays for metabolic activity, CFU counts for viability, and microscopy for growth, viability, and morphological analysis. The CAGTA-enriched IgG fraction significantly reduced the metabolic activity and viability of C. albicans compared to anti-blastospores. Purified CAGTA altered germ tube cell wall surfaces, as revealed by electron microscopy, and exhibited fungicidal properties by DiBAC fluorescent staining. In conclusion, antibodies in response to invasive candidiasis have antifungal activity against Candida albicans, influencing metabolic activity, viability, and cell wall structure, leading to cell death. These findings suggest the potential utility of CAGTA as diagnostic markers and support the possibility of developing immunization protocols against Candida infections.

Keywords: Antibodies; Candida albicans; Fungicides; Growth inhibitor; Invasive candidiasis.

Fig. 1

Scheme of the process for obtaining different serum fractions from patients with invasive candidiasis. Serum w-IgG from immune serum (1) was purified using the Melon kit. Then, it was incubated with heat-killed C. albicans yeast (2) to retain the antiBL-IgG (3) that were eluted after being centrifuged. The supernatant containing CAGTAs was termed "CAGTA enriched fraction" (CAGTA-enr IgG) (4). The pur-CAGTA (6) were eluted from the surface of C. albicans germ tubes that were incubated with the CAGTA-enr fraction (5). Abbreviations: whole IgG (w-IgG), anti-blastospores antibodies (AntiBL-IgG), CAGTA enriched fraction (CAGTA-enr IgG), and purified CAGTA (pur-CAGTA)

Fig. 2

Metabolic activity and viability of planktonic cells of C. albicans SC5314 after being exposed to different human serum IgG fractions for 2.5 h in SDB at 37 °C. Control cultures were run without antibodies or with an Irr-IgG. Metabolic activity was estimated with XTT colorimetric assay (a, b and c), and viability was referred to as CFU/ml (d). Bars represent mean value ± SD from three independent experiments. *Statistical significance was established with reference to control cells using the Kruskal–Wallis test (p < 0.05). Abbreviations: whole IgG (w-IgG), CAGTA enriched fraction (CAGTA-enr IgG), anti-blastospores antibodies (AntiBL-IgG), irrelevant IgG (Irr-IgG) and NAC (non-albicans Candida spp).

Fig. 3

Phase contrast microscopy of C. albicans SC 5314 grown at 37 °C for 2.5 h in SBD supplemented with different concentrations of an Irr-IgG or CAGTA-enr IgG. CTRL were incubated without antibodies. Bar: 20 μm. Abbreviations: CAGTA enriched fraction (CAGTA-enr IgG), irrelevant IgG (Irr-IgG) and control cells (CTRL)

Fig. 4

SEM images of C. albicans SC5314 blastospores (BL) and mycelia (M) grown at 37 °C for 2.5 h in SDB with pur-CAGTA 20 μg/ml (b, d, f, h, j) or without antibodies (a, c, e, g, i). Magnification: × 20,000 (a, b, e, f) and × 50,000 (c, d, g–j) for details. Arrows highlight surface protuberances. Abbreviations: blastospore (BL) and mycelia (M), purified CAGTA (pur-CAGTA)

Fig. 5

Photomicrographs of C. albicans SC5314 grown at 37 °C for 2.5 h in SBD with pur-CAGTA 20 μg/ml and then stained with the fluorescent dyes CFDA and DiBAC. Staining control consisted of cells grown in SBD without antibodies live and heat-killed. Paired images depict epifluorescent (on the left) and phase contrast microscopy (on the right) of the same field. Bar: 20 μm. Abbreviations: purified CAGTA (pur-CAGTA)